Ultra bright dimeric or polymeric dyes with rigid spacing groups

ABSTRACT

Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I) or a stereoisomer, tautomer or salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , L 5 , A, M, m and n are as defined herein. Methods associated with preparation and use of such compounds are also provided.

BACKGROUND Field

The present invention is generally directed to dimeric and polymericfluorescent or colored dyes having rigid spacing groups, and methods fortheir preparation and use in various analytical methods.

Description of the Related Art

Fluorescent and/or colored dyes are known to be particularly suitablefor applications in which a highly sensitive detection reagent isdesirable. Dyes that are able to preferentially label a specificingredient or component in a sample enable the researcher to determinethe presence, quantity and/or location of that specific ingredient orcomponent. In addition, specific systems can be monitored with respectto their spatial and temporal distribution in diverse environments.

Fluorescence and colorimetric methods are extremely widespread inchemistry and biology. These methods give useful information on thepresence, structure, distance, orientation, complexation and/or locationfor biomolecules. In addition, time-resolved methods are increasinglyused in measurements of dynamics and kinetics. As a result, manystrategies for fluorescence or color labeling of biomolecules, such asnucleic acids and protein, have been developed. Since analysis ofbiomolecules typically occurs in an aqueous environment, the focus hasbeen on development and use of water soluble dyes.

Highly fluorescent or colored dyes are desirable since use of such dyesincreases the signal to noise ratio and provides other related benefits.Accordingly, attempts have been made to increase the signal from knownfluorescent and/or colored moieties. For example, dimeric and polymericcompounds comprising two or more fluorescent and/or colored moietieshave been prepared in anticipation that such compounds would result inbrighter dyes. However, as a result of intramolecular fluorescencequenching, the known dimeric and polymeric dyes have not achieved thedesired increase in brightness.

There is thus a need in the art for water soluble dyes having anincreased molar brightness. Ideally, such dyes and biomarkers should beintensely colored or fluorescent and should be available in a variety ofcolors and fluorescent wavelengths. The present invention fulfills thisneed and provides further related advantages.

BRIEF SUMMARY

In brief, embodiments of the present invention are generally directed tocompounds useful as water soluble, fluorescent and/or colored dyesand/or probes that enable visual detection of analyte molecules, such asbiomolecules, as well as reagents for their preparation. Methods forvisually detecting analyte molecules using the dyes are also described.

Embodiments of the presently disclosed dyes include two or morefluorescent and/or colored moieties covalently linked by a rigid linker(“A”). In contrast to previous reports of dimeric and/or polymeric dyes,the present dyes are significantly brighter than the correspondingmonomeric dye compound. While, not wishing to be bound by theory, it isbelieved that the rigid linker moiety provides sufficient spatialseparation between the fluorescent and/or colored moieties such thatintramolecular fluorescence quenching is reduced and/or eliminated.

The water soluble, fluorescent or colored dyes of embodiments of theinvention are intensely colored and/or fluorescent and can be readilyobserved by visual inspection or other means. In some embodiments thecompounds may be observed without prior illumination or chemical orenzymatic activation. By appropriate selection of the dye, as describedherein, visually detectable analyte molecules of a variety of colors maybe obtained.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, tautomer or salt thereof, wherein R¹, R², R³, R⁴, R⁵,L¹, L², L³, L⁴, L⁵, A, M, m and n are as defined herein. Compounds ofstructure (I) find utility in a number of applications, including use asfluorescent and/or colored dyes in various analytical methods.

In another embodiment, a method for staining a sample is provided, themethod comprises adding to said sample a compound of structure (I) in anamount sufficient to produce an optical response when said sample isilluminated at an appropriate wavelength.

In still other embodiments, the present disclosure provides a method forvisually detecting an analyte molecule, comprising:

(a) providing a compound of (I); and

(b) detecting the compound by its visible properties.

Other disclosed methods include a method for visually detecting abiomolecule, the method comprising:

(a) admixing a compound of structure (I) with one or more biomolecules;and

(b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising a compound ofstructure (I) and one or more biomolecules. Use of such compositions inanalytical methods for detection of the one or more biomolecules is alsoprovided.

In some other different embodiments is provided a compound of structure(II):

or a stereoisomer, salt or tautomer thereof, wherein R¹, R², R³, R⁴, R⁵,L^(1a), L², L³, L⁴, L⁵, A, G, m and n are as defined herein. Compoundsof structure (II) find utility in a number of applications, includinguse as intermediates for preparation of fluorescent and/or colored dyesof structure (I).

In yet other embodiments a method for labeling an analyte molecule isprovided, the method comprising:

(a) admixing a compound of structure (II), wherein R² or R³ is Q or alinker comprising a covalent bond to Q, with the analyte molecule;

(b) forming a conjugate of the compound and the analyte molecule; and

(c) reacting the conjugate with a compound of formula M-L^(1b)-G′,thereby forming at least one covalent bond by reaction of G and G′,wherein R², R³, Q, G and M-L^(1b)-G′ are as defined herein.

In some different embodiments another method for labeling an analytemolecule is provided, the method comprising:

(a) admixing a compound of structure (II), wherein R² or R³ is Q or alinker comprising a covalent bond to Q, with a compound of formulaM-L^(1b)-G′, thereby forming at least one covalent bond by reaction of Gand G; and

(b) reacting the product of step (A) with the analyte molecule, therebyforming a conjugate of the product of step (A) and the analyte moleculewherein R², R³, Q, G and M-L^(1b)-G′ are as defined herein.

In more different embodiments, a method for preparing a compound ofstructure (I) is provided, the method comprising admixing a compound ofstructure (II) with a compound of formula M-L^(1b)-G′, thereby formingat least one covalent bond by reaction of G and G′, wherein G andM-L^(1b)-G′ are as defined herein.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 provides UV absorbance spectra for comparative dye compounds.

FIG. 2 provides fluorescence emission spectra for comparative dyecompounds.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ group.

“Carboxy” refers to the —CO₂H group.

“Cyano” refers to the —CN group.

“Formyl” refers to the —C(═O)H group.

“Hydroxy” or “hydroxyl” refers to the —OH group.

“Imino” refers to the ═NH group.

“Nitro” refers to the —NO₂ group.

“Oxo” refers to the ═O substituent group.

“Sulfhydryl” refers to the —SH group.

“Thioxo” refers to the ═S group.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to twelve carbon atoms (C₁-C₁₂ alkyl), oneto eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, and the like. Unless stated otherwise specifically in thespecification, alkyl groups are optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, containing no unsaturation,and having from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single bond and to the radicalgroup through a single bond. The points of attachment of the alkylenechain to the rest of the molecule and to the radical group can bethrough one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, alkylene is optionallysubstituted.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon double bond and having from two to twelve carbon atoms,e.g., ethenylene, propenylene, n-butenylene, and the like. Thealkenylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkenylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, alkenylene is optionally substituted.

“Alkynylene” or “alkynylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon triple bond and having from two to twelve carbon atoms,e.g., ethenylene, propenylene, n-butenylene, and the like. Thealkynylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkynylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, alkynylene is optionally substituted.

“Alkylether” refers to any alkyl group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylether. Unless stated otherwise specifically in the specification,an alkylether group is optionally substituted. For example, in someembodiments an alkylether is substituted with an alcohol or—OP(═R_(a))(R_(b))R_(c), wherein each of R_(a), R_(b) and R_(c) is asdefined for compounds of structure (I).

“Alkoxy” refers to a group of the formula —OR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup is optionally substituted.

“Heteroalkylene” refers to an alkylene group, as defined above,comprising at least one heteroatom (e.g., N, O, P or S) within thealkylene chain or at a terminus of the alkylene chain. In someembodiments, the heteroatom is within the alkylene chain (i.e., theheteroalkylene comprises at least one carbon-heteroatom-carbon bond). Inother embodiments, the heteroatom is at a terminus of the alkylene andthus serves to join the alkylene to the remainder of the molecule (e.g.,M1-H-B-M2, where M1 and M2 are portions of the molecule, H is aheteroatom and B is an alkylene). Unless stated otherwise specificallyin the specification, a heteroalkylene group is optionally substituted.An exemplary heteroalkylene linking group is illustrated below:

Multimers of the above C-linker are included in various embodiments ofheteroalkylene linkers.

“Heteroalkenylene” is a heteroalkylene, as defined above, comprising atleast one carbon-carbon double bond. Unless stated otherwisespecifically in the specification, a heteroalkenylene group isoptionally substituted.

“Heteroalkynylene” is a heteroalkylene comprising at least onecarbon-carbon triple bond. Unless stated otherwise specifically in thespecification, a heteroalkynylene group is optionally substituted.

“Heteroatomic” in reference to a “heteroatomic linker” refers to alinker group consisting of one or more heteroatom. Exemplaryheteroatomic linkers include single atoms selected from the groupconsisting of O, N, P and S, and multiple heteroatoms for example alinker having the formula —P(O⁻)(═O)O— or —OP(O⁻)(═O)O— and multimersand combinations thereof.

“Phosphate” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a thiophosphate group ora further phosphate group, wherein R_(c) is a counter ion (e.g., Na+ andthe like).

“Phosphoalkyl” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is —Oalkyl, wherein R_(c) is a counter ion(e.g., Na+ and the like). Unless stated otherwise specifically in thespecification, a phosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in a phosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether.

“Phosphoalkylether” refers to the —OP(═O)(R_(a))R_(b) group, whereinR_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylether, wherein R_(c) is acounter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphoalkylether group isoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a phosphoalkylether group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether.

“Thiophosphate” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH,SH, O⁻, S⁻, OR_(d), SR_(d), a phosphate group or a further thiophosphategroup, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: i) R_(a) is S; ii) R_(b) is S″ or SR_(d); iii) R_(c) isSH, S⁻ or SR_(d); or iv) a combination of i), ii) and/or iii).

“Thiophosphoalkyl” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is—Oalkyl, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: i) R_(a) is S; ii) R_(b) is S″ or SR_(d); or iii)R_(a) isS and R_(b) is S⁻ or SR_(d). Unless stated otherwise specifically in thespecification, a thiophosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in athiophosphoalkyl group is optionally substituted with one or more ofhydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.

“Thiophosphoalkylether” refers to the —OP(═R_(a))(R_(b))R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and is—Oalkylether, wherein R_(d) is a counter ion (e.g., Na+ and the like)and provided that: i) R_(a) is S; ii) R_(b) is S″ or SR_(d); oriii)R_(a) is S and R_(b) is S″ or SR_(d). Unless stated otherwisespecifically in the specification, a thiophosphoalkylether group isoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a thiophosphoalkyl group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether.

“Carbocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising 3 to 18 carbon atoms. Unless statedotherwise specifically in the specification, a carbocyclic ring may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems, and may be partially or fullysaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, whilearomatic carbocyclyl radicals include aryl. Unless stated otherwisespecifically in the specification, a carbocyclic group is optionallysubstituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycycliccarbocyclic ring, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic cyclocalkylsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptly, and cyclooctyl. Polycyclic cycloalkyls include, forexample, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless statedotherwise specifically in the specification, a cycloalkyl group isoptionally substituted.

“Aryl” refers to a ring system comprising at least one carbocyclicaromatic ring. In some embodiments, an aryl comprises from 6 to 18carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryls include, but are not limited to, aryls derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, an aryl group is optionally substituted.

“Heterocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising one to twelve carbon atoms and from one tosix heteroatoms selected from the group consisting of nitrogen, oxygenand sulfur. Unless stated otherwise specifically in the specification,the heterocyclic ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclicring may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclic ring may be partially or fullysaturated. Examples of aromatic heterocyclic rings are listed below inthe definition of heteroaryls (i.e., heteroaryl being a subset ofheterocyclic). Examples of non-aromatic heterocyclic rings include, butare not limited to, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl,trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl,thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.Unless stated otherwise specifically in the specification, aheterocyclic group is optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system comprising one tothirteen carbon atoms, one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and at least one aromaticring. For purposes of certain embodiments of this invention, theheteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,benzoxazolinonyl, benzimidazolthionyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl,imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl,1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pryrimidinonyl,pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl,quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl,thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl,tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless statedotherwise specifically in the specification, a heteroaryl group isoptionally substituted.

“Fused” refers to a ring system comprising at least two rings, whereinthe two rings share at least one common ring atom, for example twocommon ring atoms. When the fused ring is a heterocyclyl ring or aheteroaryl ring, the common ring atom(s) may be carbon or nitrogen.Fused rings include bicyclic, tricyclic, tertracyclic, and the like.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, heteroalkynylene, alkoxy, alkylether, phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether, carbocyclic,cycloalkyl, aryl, heterocyclic and/or heteroaryl) wherein at least onehydrogen atom (e.g., 1, 2, 3 or all hydrogen atoms) is replaced by abond to a non-hydrogen atoms such as, but not limited to: a halogen atomsuch as F, Cl, Br, and I; an oxygen atom in groups such as hydroxylgroups, alkoxy groups, and ester groups; a sulfur atom in groups such asthiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, andsulfoxide groups; a nitrogen atom in groups such as amines, amides,alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, and enamines; a silicon atom in groups such astrialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups,and triarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g),—SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h). 37Substituted also means any of the above groups in which one or morehydrogen atoms are replaced with —C(═O)R_(g), —C(═O)OR_(g),—C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). In the foregoing,R_(g) and R_(h) are the same or different and independently hydrogen,alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl.“Substituted” further means any of the above groups in which one or morehydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl,imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl,aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl group. In addition, each of the foregoing substituentsmay also be optionally substituted with one or more of the abovesubstituents.

“Conjugation” refers to the overlap of one p-orbital with anotherp-orbital across an intervening sigma bond. Conjugation may occur incyclic or acyclic compounds. A “degree of conjugation” refers to theoverlap of at least one p-orbital with another p-orbital across anintervening sigma bond. For example, 1, 3-butadine has one degree ofconjugation, while benzene and other aromatic compounds typically havemultiple degrees of conjugation. Fluorescent and colored compoundstypically comprise at least one degree of conjugation.

“Fluorescent” refers to a molecule which is capable of absorbing lightof a particular frequency and emitting light of a different frequency.Fluorescence is well-known to those of ordinary skill in the art.

“Colored” refers to a molecule which absorbs light within the coloredspectrum (i.e., red, yellow, blue and the like).

A “linker” refers to a contiguous chain of at least one atom, such ascarbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof,which connects a portion of a molecule to another portion of the samemolecule or to a different molecule, moiety or solid support (e.g.,microparticle). Linkers may connect the molecule via a covalent bond orother means, such as ionic or hydrogen bond interactions.

The term “biomolecule” refers to any of a variety of biologicalmaterials, including nucleic acids, carbohydrates, amino acids,polypeptides, glycoproteins, hormones, aptamers and mixtures thereof.More specifically, the term is intended to include, without limitation,RNA, DNA, oligonucleotides, modified or derivatized nucleotides,enzymes, receptors, prions, receptor ligands (including hormones),antibodies, antigens, and toxins, as well as bacteria, viruses, bloodcells, and tissue cells. The visually detectable biomolecules of theinvention (e.g., compounds of structure (I) having a biomolecule linkedthereto) are prepared, as further described herein, by contacting abiomolecule with a compound having a reactive group that enablesattachment of the biomolecule to the compound via any available atom orfunctional group, such as an amino, hydroxy, carboxyl, or sulfhydrylgroup on the biomolecule.

A “reactive group” is a moiety capable of reacting with a secondreactive groups (e.g., a “complementary reactive group”) to form one ormore covalent bonds, for example by a displacement, oxidation,reduction, addition or cycloaddition reaction. Exemplary reactive groupsare provided in Table 1, and include for example, nucleophiles,electrophiles, dienes, dienophiles, aldehyde, oxime, hydrazone, alkyne,amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl,disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester,ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide,epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, thiiraneand the like.

The terms “visible” and “visually detectable” are used herein to referto substances that are observable by visual inspection, without priorillumination, or chemical or enzymatic activation. Such visuallydetectable substances absorb and emit light in a region of the spectrumranging from about 300 to about 900 nm. Preferably, such substances areintensely colored, preferably having a molar extinction coefficient ofat least about 40,000, more preferably at least about 50,000, still morepreferably at least about 60,000, yet still more preferably at leastabout 70,000, and most preferably at least about 80,000M⁻¹ cm⁻¹. Thecompounds of the invention may be detected by observation with the nakedeye, or with the aid of an optically based detection device, including,without limitation, absorption spectrophotometers, transmission lightmicroscopes, digital cameras and scanners. Visually detectablesubstances are not limited to those which emit and/or absorb light inthe visible spectrum. Substances which emit and/or absorb light in theultraviolet (UV) region (about 10 nm to about 400 nm), infrared (IR)region (about 700 nm to about 1 mm), and substances emitting and/orabsorbing in other regions of the electromagnetic spectrum are alsoincluded with the scope of “visually detectable” substances.

For purposes of embodiments of the invention, the term “photostablevisible dye” refers to a chemical moiety that is visually detectable, asdefined hereinabove, and is not significantly altered or decomposed uponexposure to light. Preferably, the photostable visible dye does notexhibit significant bleaching or decomposition after being exposed tolight for at least one hour. More preferably, the visible dye is stableafter exposure to light for at least 12 hours, still more preferably atleast 24 hours, still yet more preferably at least one week, and mostpreferably at least one month. Nonlimiting examples of photostablevisible dyes suitable for use in the compounds and methods of theinvention include azo dyes, thioindigo dyes, quinacridone pigments,dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole,quinophthalone, and truarycarbonium.

As used herein, the term “perylene derivative” is intended to includeany substituted perylene that is visually detectable. However, the termis not intended to include perylene itself. The terms “anthracenederivative”, “naphthalene derivative”, and “pyrene derivative” are usedanalogously. In some preferred embodiments, a derivative (e.g.,perylene, pyrene, anthracene or naphthalene derivative) is an imide,bisimide or hydrazamimide derivative of perylene, anthracene,naphthalene, or pyrene.

The visually detectable molecules of various embodiments of theinvention are useful for a wide variety of analytical applications, suchas biochemical and biomedical applications, in which there is a need todetermine the presence, location, or quantity of a particular analyte(e.g., biomolecule). In another aspect, therefore, the inventionprovides a method for visually detecting a biomolecule, comprising: (a)providing a biological system with a visually detectable biomoleculecomprising the compound of structure (I) linked to a biomolecule; and(b) detecting the biomolecule by its visible properties. For purposes ofthe invention, the phrase “detecting the biomolecule by its visibleproperties” means that the biomolecule, without illumination or chemicalor enzymatic activation, is observed with the naked eye, or with the aidof a optically based detection device, including, without limitation,absorption spectrophotometers, transmission light microscopes, digitalcameras and scanners. A densitometer may be used to quantify the amountof visually detectable biomolecule present. For example, the relativequantity of the biomolecule in two samples can be determined bymeasuring relative optical density. If the stoichiometry of dyemolecules per biomolecule is known, and the extinction coefficient ofthe dye molecule is known, then the absolute concentration of thebiomolecule can also be determined from a measurement of opticaldensity. As used herein, the term “biological system” is used to referto any solution or mixture comprising one or more biomolecules inaddition to the visually detectable biomolecule. Nonlimiting examples ofsuch biological systems include cells, cell extracts, tissue samples,electrophoretic gels, assay mixtures, and hybridization reactionmixtures.

“Solid support” refers to any solid substrate known in the art forsolid-phase support of molecules, for example a “microparticle” refersto any of a number of small particles useful for attachment to compoundsof the invention, including, but not limited to, glass beads, magneticbeads, polymeric beads, nonpolymeric beads, and the like. In certainembodiments, a microparticle comprises polystyrene beads.

“Base pairing moiety” refers to a heterocyclic moiety capable ofhybridizing with a complementary heterocyclic moiety via hydrogen bonds(e.g., Watson-Crick base pairing). Base pairing moieties include naturaland unnatural bases. Non-limiting examples of base pairing moieties areRNA and DNA bases such adenosine, guanosine, thymidine, cytosine anduridine and analogues thereof.

Embodiments of the invention disclosed herein are also meant toencompass all compounds of structure (I) or (II) beingisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, 35S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively.

Isotopically-labeled compounds of structure (I) or (II) can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described below and in the followingExamples using an appropriate isotopically-labeled reagent in place ofthe non-labeled reagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted alkyl” means that thealkyl group may or may not be substituted and that the descriptionincludes both substituted alkyl groups and alkyl groups having nosubstitution.

“Salt” includes both acid and base addition salts.

“Acid addition salt” refers to those salts which are formed withinorganic acids such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Base addition salt” refers to those salts which are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asammonia, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Crystallizations may produce a solvate of the compounds describedherein. Embodiments of the present invention include all solvates of thedescribed compounds. As used herein, the term “solvate” refers to anaggregate that comprises one or more molecules of a compound of theinvention with one or more molecules of solvent. The solvent may bewater, in which case the solvate may be a hydrate. Alternatively, thesolvent may be an organic solvent. Thus, the compounds of the presentinvention may exist as a hydrate, including a monohydrate, dihydrate,hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, aswell as the corresponding solvated forms. The compounds of the inventionmay be true solvates, while in other cases the compounds of theinvention may merely retain adventitious water or another solvent or bea mixture of water plus some adventitious solvent.

Embodiments of the compounds of the invention (e.g., compounds ofstructure I or II), or their salts, tautomers or solvates may containone or more asymmetric centers and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)-for amino acids. Embodiments of the present invention are meant toinclude all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds. Various tautomeric forms of thecompounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0software naming program (CambridgeSoft). Common names familiar to one ofordinary skill in the art are also used.

As noted above, in one embodiment of the present invention, compoundsuseful as fluorescent and/or colored dyes in various analytical methodsare provided. In other embodiments, compounds useful as syntheticintermediates for preparation of compounds useful as fluorescent and/orcolored dyes are provided. In general terms, embodiments of the presentinvention are directed to dimers and higher polymers of fluorescentand/or colored moieties. The fluorescent and or colored moieties arelinked by one or more rigid linkers (i.e., moiety “A”). Without wishingto be bound by theory, it is believed the linker helps to maintainsufficient spatial distance between the fluorescent and/or coloredmoieties such that intramolecular quenching is reduced or eliminated,thus resulting in a dye compound having a high molar “brightness” (e.g.,high fluorescence emission).

Accordingly, in some embodiments the compounds have the followingstructure (A):

wherein L is a linker with sufficient rigidity to maintain spatialseparation between one or more (e.g., each) M group so thatintramolecular quenching is reduced or eliminated, and R¹, R², R³, L¹,L², L³ and n are as defined for structure (I).

In other embodiments is provided a compound having the followingstructure (I):

or a stereoisomer, salt or tautomer thereof, wherein:

A is, at each occurrence, independently a moiety comprising one or more,fused, carbocyclic or heterocyclic ring system;

M is, at each occurrence, independently a moiety comprising two or morecarbon-carbon double bonds and at least one degree of conjugation;

L¹ is at each occurrence, independently either: i) an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker; or ii) a linker comprising afunctional group capable of formation by reaction of two complementaryreactive groups;

L², L³, L⁴ and L⁵ are, at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene linker or heteroatomic linker;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,—OP(═R_(a))(R_(b))R_(c), Q, a linker comprising a covalent bond to Q, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support or a linker comprising acovalent bond to a further compound of structure (I), wherein: R_(a) isO or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R_(c) is OH, SH, O⁻,S⁻, OR_(d), SR_(d), alkyl, alkoxy, alkylether, alkoxyalkylether,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether; and R_(d) is a counter ion;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with an analyte molecule, asolid support or a complementary reactive group Q;

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and

n is an integer of one or greater.

The various linkers and substituents (e.g., M, A, Q, R¹, R², R³, R^(c)L¹, L², L³, L⁴ and L⁵) in the compound of structure (I) are optionallysubstituted with one more substituent. For example, in some embodimentsthe optional substituent is selected to optimize the water solubility orother property of the compound of structure (I). In certain embodiments,each alkyl, alkoxy, alkylether, alkoxyalkylether, phosphoalkyl,thiophosphoalkyl, phosphoalkylether and thiophosphoalkylether in thecompound of structure (I) is optionally substituted with one moresubstituent selected from the group consisting of hydroxyl, alkoxy,alkylether, alkoxyalkylether, sulfhydryl, amino, alkylamino, carboxyl,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether and thiophosphoalkylether.

In some embodiments, A is at each occurrence, independently a moietycomprising one or more, fused, aryl or heteroaryl ring system. Indifferent embodiments, A is at each occurrence, independently a moietycomprising one or more, fused, bicyclic or tricyclic, aryl or heteroarylring system.

In other more specific embodiments, A is, at each occurrence,independently a fused, carbocyclic or heterocyclic ring system havingone of the following structures:

wherein:

a¹, a² and a³ are, at each occurrence, independently a 5, 6 or7-membered carbocyclic or heterocyclic ring; and

L⁶ is a direct bond or a linker.

In yet other embodiments A, at each occurrence, independently has one ofthe following structures:

The optional linker L¹ can be used as a point of attachment of the Mmoiety to the remainder of the compound. For example, in someembodiments a synthetic precursor to the compound of structure (I) isprepared, and the M moiety is attached to the synthetic precursor usingany number of facile methods known in the art, for example methodsreferred to as “click chemistry.” For this purpose any reaction which israpid and substantially irreversible can be used to attach M to thesynthetic precursor to form a compound of structure (I). Exemplaryreactions include the copper catalyzed reaction of an azide and alkyneto form a triazole (Huisgen 1, 3-dipolar cycloaddition), reaction of adiene and dienophile (Diels-Alder), strain-promoted alkyne-nitronecycloaddition, reaction of a strained alkene with an azide, tetrazine ortetrazole, alkene and azide [3+2] cycloaddition, alkene and tetrazineinverse-demand Diels-Alder, alkene and tetrazole photoreaction andvarious displacement reactions, such as displacement of a leaving groupby nucleophilic attack on an electrophilic atom. In some embodiments thereaction to form L¹ may be performed in an aqueous environment.

Accordingly, in some embodiments L¹ is at each occurrence a linkercomprising a functional group capable of formation by reaction of twocomplementary reactive groups, for example a functional group which isthe product of one of the foregoing “click” reactions. In variousembodiments, for at least one occurrence of L¹, the functional group canbe formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine,azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide,sulfonyl halide, isothiocyanate, imidoester, activated ester, ketone,α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide,aziridine, tetrazine, tetrazole, phosphine, biotin or thiiranefunctional group with a complementary reactive group.

In other embodiments, for at least one occurrence of L¹, the functionalgroup can be formed by reaction of an alkyne and an azide.

In more embodiments, for at least one occurrence of L¹, the functionalgroup comprises an alkene, ester, amide, thioester, disulfide,carbocyclic, heterocyclic or heteroaryl group. In some more specificembodiments, for at least one occurrence of L¹, L¹ is a linkercomprising a triazolyl functional group.

In still other embodiments, for at least one occurrence of L¹, L¹-M hasthe following structure:

wherein L^(1a) and L^(1b) are each independently optional linkers.

In different embodiments, for at least one occurrence of L¹, L¹-M hasthe following structure:

wherein L^(1a) and L^(1b) are each independently optional linkers.

In various embodiments of the foregoing, L^(1a) or L^(1b), or both, isabsent. In other embodiments, L^(1a) or L^(1b), or both, is present.

In some embodiments L^(1a) and L^(1b), when present, are eachindependently alkylene or heteroalkylene. For example, in someembodiments L^(1a) and L^(1b), when present, independently have one ofthe following structures:

In still other different embodiments of structure (I), L¹ is at eachoccurrence, independently an optional alkylene or heteroalkylene linker.

In more embodiments, L², L³, L⁴ and L⁵ are, at each occurrence,independently C₁-C₆ alkylene, C₂-C₆ alkenylene or C₂-C₆ alkynylene. Forexample, in some embodiments the compound has the following structure(IA):

wherein:

x¹, x², x³, x⁴, x⁵ and x⁶ are, at each occurrence, independently aninteger from 0 to 6.

In certain embodiments of the compound of structure (IA), x³ and x⁴ areboth 2 at each occurrence. In other embodiments, x¹, x², x⁵ and x⁶ areeach 1 at each occurrence.

In some more specific embodiments of the compound of structure (IA), L¹,at each occurrence, independently comprises a triazolyl functionalgroup. In other embodiments of the compound of structure (IA), L¹, ateach occurrence, independently an optional alkylene or heteroalkylenelinker.

In still other embodiments of any of the compounds of structure (I) or(IA), R⁴ is, at each occurrence, independently OH, O⁻ or OR_(d). It isunderstood that “OR_(d)” and “SR_(d)” are intended to refer to O⁻ and S⁻associated with a cation. For example, the disodium salt of a phosphategroup may be represented as:

where R_(a) is sodium (Na⁺).

In other embodiments of any of the compounds of structure (I) or (IA),R⁵ is, at each occurrence, oxo.

In some different embodiments of any of the foregoing compounds, R¹ isH.

In other various embodiments, R² and R³ are each independently OH or—OP(═R_(a))(R_(b))R_(c). In some different embodiments, R² or R³ is OHor —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q.

In still other embodiments, Q is, at each occurrence, independently amoiety comprising a reactive group capable of forming a covalent bondwith an analyte molecule or a solid support. In other embodiments, Q is,at each occurrence, independently a moiety comprising a reactive groupcapable of forming a covalent bond with a complementary reactive groupQ′. For example, in some embodiments, Q′ is present on a furthercompound of structure (I) (e.g., in the R² or R³ position), and Q and Q′comprise complementary reactive groups such that reaction of thecompound of structure (I) and the further compound of structure (I)results in covalently bound dimer of the compound of structure (I).Multimer compounds of structure (I) can also be prepared in an analogousmanner and are included within the scope of embodiments of theinvention.

The type of Q group and connectivity of the Q group to the remainder ofthe compound of structure (I) is not limited, provided that Q comprisesa moiety having appropriate reactivity for forming the desired bond.

In certain embodiments, the Q is a moiety which is not susceptible tohydrolysis under aqueous conditions, but is sufficiently reactive toform a bond with a corresponding group on an analyte molecule or solidsupport (e.g., an amine, azide or alkyne).

Certain embodiments of compounds of structure (I) comprises Q groupscommonly employed in the field of bioconjugation. For example in someembodiments, Q comprises a nucleophilic reactive group, an electrophilicreactive group or a cycloaddition reactive group. In some more specificembodiments, Q comprises a sulfhydryl, disulfide, activated ester,isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide,sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimidefunctional group. In some embodiments, the activated ester is anN-succinimide ester, imidoester or polyflourophenyl ester. In otherembodiments, the alkyne is an alkyl azide or acyl azide.

Exemplary Q moieties are provided in Table I below.

TABLE 1 Exemplary Q Moieties Structure Class

Sulfhydryl

Isothio- cyanate

Imidoester

Acyl Azide

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Sulfonyl halide

Maleimide

Maleimide

α- haloimide

Disulfide

Phosphine

Azide

Alkyne

Biotin

Diene

Alkene/ dienophile

Alkene/ dienophile —NH₂ Amino

It should be noted that in some embodiments, wherein Q is SH, the SHmoiety will tend to form disulfide bonds with another sulfhydryl groupon another compound of structure (I). Accordingly, some embodimentsinclude compounds of structure (I), which are in the form of disulfidedimers, the disulfide bond being derived from SH Q groups.

In some other embodiments, one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is a linkercomprising a covalent bond to an analyte molecule or a linker comprisinga covalent bond to a solid support. For example, in some embodiments theanalyte molecule is a nucleic acid, amino acid or a polymer thereof. Inother embodiments, the analyte molecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion. In still differentembodiments, the solid support is a polymeric bead or nonpolymeric bead.

The value for m is another variable that can be selected based on thedesired fluorescence and/or color intensity. In some embodiments, m is,at each occurrence, independently an integer of 2 or more. In someembodiments, m is, at each occurrence, independently an integer from 1to 10, 3 to 10 or 3 to 6. In other embodiments, m is, at eachoccurrence, independently an integer from 1 to 5, for example 1, 2, 3, 4or 5. In other embodiments, m is, at each occurrence, independently aninteger from 5 to 10, for example 5, 6, 7, 8, 9 or 10.

The fluorescence intensity can also be tuned by selection of differentvalues of n. In certain embodiments, n is an integer from 1 to 100. Inother embodiments, n is an integer from 1 to 10. In some embodiments, nis 1.

M is selected based on the desired optical properties, for example basedon a desired color and/or fluorescence emission wavelength. In someembodiments, M is the same at each occurrence; however, it is importantto note that each occurrence of M need not be an identical M, andcertain embodiments include compounds wherein M is not the same at eachoccurrence. For example, in some embodiments each M is not the same andthe different M moieties are selected to have absorbance and/oremissions for use in fluorescence resonance energy transfer (FRET)methods. For example, in such embodiments the different M moieties areselected such that absorbance of radiation at one wavelength causesemission of radiation at a different wavelength by a FRET mechanism.Exemplary M moieties can be appropriately selected by one of ordinaryskill in the art based on the desired end use. Exemplary M moieties forFRET methods include fluorescein and 5-TAMRA(5-carboxytetramethylrhodamine, succinimidyl ester) dyes.

M may be attached to the remainder of the molecule from any position(i.e., atom) on M. One of skill in the art will recognize means forattaching M to the remainder of molecule. Exemplary methods include the“click” reactions described herein.

In some embodiments, M is a fluorescent or colored moiety. Anyfluorescent and/or colored moiety may be used, for examples those knownin the art and typically employed in colorimetric, UV, and/orfluorescent assays may be used. Examples of M moieties which are usefulin various embodiments of the invention include, but are not limited to:Xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosinor Texas red); Cyanine derivatives (e.g., cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine or merocyanine); Squaraine derivativesand ring-substituted squaraines, including Seta, SeTau, and Square dyes;Naphthalene derivatives (e.g., dansyl and prodan derivatives); Coumarinderivatives; oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole or benzoxadiazole); Anthracene derivatives (e.g.,anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); Pyrenederivatives such as cascade blue; Oxazine derivatives (e.g., Nile red,Nile blue, cresyl violet, oxazine 170); Acridine derivatives (e.g.,proflavin, acridine orange, acridine yellow); Arylmethine derivatives:auramine, crystal violet, malachite green; and Tetrapyrrole derivatives(e.g., porphin, phthalocyanine or bilirubin). Other exemplary M moietiesinclude: Cyanine dyes, xanthate dyes (e.g., Hex, Vic, Nedd, Joe or Tet);Yakima yellow; Redmond red; tamra; texas red and Alexa Fluor® dyes.

In still other embodiments of any of the foregoing, M comprises three ormore aryl or heteroaryl rings, or combinations thereof, for example fouror more aryl or heteroaryl rings, or combinations thereof, or even fiveor more aryl or heteroaryl rings, or combinations thereof. In someembodiments, M comprises six aryl or heteroaryl rings, or combinationsthereof. In further embodiments, the rings are fused. For example insome embodiments, M comprises three or more fused rings, four or morefused rings, five or more fused rings, or even six or more fused rings.

In some embodiments, M is cyclic. For example, in some embodiments M iscarbocyclic. In other embodiment, M is heterocyclic. In still otherembodiments of the foregoing, M, at each occurrence, independentlycomprises an aryl moiety. In some of these embodiments, the aryl moietyis multicyclic. In other more specific examples, the aryl moiety is afused-multicyclic aryl moiety, for example which may comprise at least3, at least 4, or even more than 4 aryl rings.

In other embodiments of any of the foregoing compounds of structure (I)or (IA), M, at each occurrence, independently comprises at least oneheteroatom. For example, in some embodiments, the heteroatom isnitrogen, oxygen or sulfur.

In still more embodiments of any of the foregoing, M, at eachoccurrence, independently comprises at least one substituent. Forexample, in some embodiments the substituent is a fluoro, chloro, bromo,iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy,aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl,carboxy, sulfonate, amide, or formyl group.

In some even more specific embodiments of the foregoing, M, at eachoccurrence, independently is a dimethylaminostilbene, quinacridone,fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine,terrylene, sexiphenyl, porphyrin, benzopyrene,(fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9, 10-ethynylanthracene or ter-naphthyl moiety.In other embodiments, M is, at each occurrence, independentlyp-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine,thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, orperylene amide or a derivative thereof. In still more embodiments, M is,at each occurrence, independently a coumarin dye, resorufin dye,dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energytransfer dye, thiazole orange dye, polymethine orN-aryl-1,8-naphthalimide dye.

In still more embodiments of any of the foregoing, M at each occurrenceis the same. In other embodiments, each M is different. In still moreembodiments, one or more M is the same and one or more M is different.

In some embodiments, M is pyrene, perylene, perylene monoimide or 6-FAMor derivative thereof. In some other embodiments, M has one of thefollowing structures:

In some specific embodiments, the compound is a compound selected fromTable 2:

TABLE 2 Exemplary Compounds of Structure I No. Structure I-1

I-2

I-3

I-4

I-5

I-6

I-7

As used in Table 2, and throughout the application, F, E and Y refer tofluorescein, perylene and pyrene moieties, respectively, and have thefollowing structures:

The presently disclosed dye compounds are “tunable,” meaning that byproper selection of the variables in any of the foregoing compounds, oneof skill in the art can arrive at a compound having a desired and/orpredetermined molar fluorescence (molar brightness). The tunability ofthe compounds allows the user to easily arrive at compounds having thedesired fluorescence and/or color for use in a particular assay or foridentifying a specific analyte of interest. Although all variables mayhave an effect on the molar fluorescence of the compounds, properselection of M, A, m and n is believed to play an important role in themolar fluorescence of the compounds. Accordingly, in one embodiment isprovided a method for obtaining a compound having a desired molarfluorescence, the method comprising selecting an M moiety having a knownfluorescence, preparing a compound of structure (I) comprising the Mmoiety, and selecting the appropriate variables for A, m and n to arriveat the desired molar fluorescence.

Molar fluorescence in certain embodiments can be expressed in terms ofthe fold increase or decrease relative to the fluorescence emission ofthe parent fluorophore (e.g., monomer). In some embodiments the molarfluorescence of the present compounds is 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×,7×, 8×, 9× 10× or even higher relative to the parent fluorophore.Various embodiments include preparing compounds having the desired foldincrease in fluorescence relative to the parent fluorophore by properselection of A, m and n.

For ease of illustration, various compounds comprising phosphorousmoieties (e.g., phosphate and the like) are depicted in the anionicstate (e.g., —OPO(OH)O⁻, —OPO₃ ²⁻). One of skill in the art will readilyunderstand that the charge is dependent on pH and the uncharged (e.g.,protonated or salt, such as sodium or other cation) forms are alsoincluded in the scope of embodiments of the invention.

Compositions comprising any of the foregoing compounds and one or moreanalyte molecules (e.g., biomolecules) are provided in various otherembodiments. In some embodiments, use of such compositions in analyticalmethods for detection of the one or more analyte molecules are alsoprovided.

In still other embodiments, the compounds are useful in variousanalytical methods. For example, in certain embodiments the disclosureprovides a method of staining a sample, the method comprising adding tosaid sample a compound of structure (I), for example wherein one of R²or R³ is a linker comprising a covalent bond to an analyte molecule(e.g., biomolecule) or microparticle, and the other of R² or R³ is H,OH, alkyl, alkoxy, alkylether or —OP(═R_(a))(R_(b))R_(c), in an amountsufficient to produce an optical response when said sample isilluminated at an appropriate wavelength.

In some embodiments of the foregoing methods, R² is a linker comprisinga covalent linkage to an analyte molecule, such as a biomolecule. Forexample, a nucleic acid, amino acid or a polymer thereof (e.g.,polynucleotide or polypeptide). In still more embodiments, thebiomolecule is an enzyme, receptor, receptor ligand, antibody,glycoprotein, aptamer or prion.

In yet other embodiments of the foregoing method, R² is a linkercomprising a covalent linkage to a solid support such as amicroparticle. For example, in some embodiments the microparticle is apolymeric bead or nonpolymeric bead.

In even more embodiments, said optical response is a fluorescentresponse.

In other embodiments, said sample comprises cells, and some embodimentsfurther comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishingthe fluorescence response from that of a second fluorophore havingdetectably different optical properties.

In other embodiments, the disclosure provides a method for visuallydetecting an analyte molecule, such as a biomolecule, comprising:

-   -   (a) providing a compound of structure (I), for example, wherein        one of R² or R³ is a linker comprising a covalent bond to the        analyte molecule, and the other of R² or R³ is H, OH, alkyl,        alkoxy, alkylether or —OP(═R_(a))(R_(b))R_(c); and    -   (b) detecting the compound by its visible properties.

In some embodiments the analyte molecule is a nucleic acid, amino acidor a polymer thereof (e.g., polynucleotide or polypeptide). In stillmore embodiments, the analyte molecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion.

In other embodiments, a method for visually detecting an analytemolecule, such as a biomolecule is provided, the method comprising:

-   -   (a) admixing any of the foregoing compounds with one or more        analyte molecules; and    -   (b) detecting the compound by its visible properties.

In other embodiments is provided a method for visually detecting ananalyte molecule, the method comprising:

-   -   (a) admixing the compound of claim 1, wherein R² or R³ is Q or a        linker comprising a covalent bond to Q, with the analyte        molecule;    -   (b) forming a conjugate of the compound and the analyte        molecule; and    -   (c) detecting the conjugate by its visible properties.

In some other different embodiments, the compounds of structure (I) canbe used in various for analysis of cells. For example, by use of flowcytometry, the compounds can be used to discriminate between live anddead cells, evaluate the health of cells (e.g., necrosis vs. earlyapoptitic vs. late apoptitic vs. live cell), tracking ploidy and mitosisduring the cell cycle and determining various states of cellproliferation. While not wishing to be bound by theory, it is believedthat embodiments of the compounds of structure (I) preferentially bindto postively charged moieties.

Accordingly, in some embodiments the compounds may be used in methodsfor determining the presence of non-intact cells, for example nectroticcells. For example, the presence of nectrotic cells can be determined byadmixing a sample containing cells with a compound of structure (I) andanalyzing the mixture by flow cytometry. The compound of structure (I)binds to nectrotic cells, and thus there presence is detectable underflow cytometry conditions. In contrast to other staining reagents whichrequire an amine reactive group to bind to nectrotic cells, embodimentsof the staining methods of employing compounds of structure (I) do notrequire a protein-free incubation buffer, and thus the methods are moreefficient to perform than related known methods.

In various other embodiments, the compounds can be used in relatedmethods for determing the presence of positively charged moieties inintact or non-intact cells, apoptitic bodies, depolarized membranesand/or permealized membranes.

In addition to the above methods, embodiments of the compounds ofstructure (I) find utility in various disciplines and methods, includingbut not limited to: imaging in endoscopy procedures for identificationof cancerous and other tissues; identification of necrotic tissue bypreferential binding of the compounds to dead cells; single-cell and/orsingle molecule analytical methods, for example detection ofpolynucleotides with little or no amplification; cancer imaging, forexample by conjugating a compound of structure (I) to an antibody orsugar or other moiety that preferentially binds cancer cells; imaging insurgical procedures; binding of histones for identification of variousdiseases; drug delivery, for example by replacing the M moiety in acompound of structure (I) with an active drug moiety; and/or contrastagents in dental work and other procedures, for example by preferentialbinding of the compound of structure (I) to various flora and/ororganisms.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific choice set forth herein for a R¹,R², R³, R⁴, R⁵, L¹, L², L³, L⁴, L⁵, A, M, m and/or n variable in thecompounds of structure (I), as set forth above, may be independentlycombined with other embodiments and/or variables of the compounds ofstructure (I) to form embodiments of the inventions not specifically setforth above. In addition, in the event that a list of choices is listedfor any particular R¹, R², R³, R⁴, R⁵, L¹, L², L³, L⁴, L⁵, A, M, mand/or n variable in a particular embodiment and/or claim, it isunderstood that each individual choice may be deleted from theparticular embodiment and/or claim and that the remaining list ofchoices will be considered to be within the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their salts by treatment with theappropriate inorganic or organic base or acid by methods known to oneskilled in the art. Salts of the compounds of the invention can beconverted to their free base or acid form by standard techniques.

The following Reaction Schemes illustrate exemplary methods of makingcompounds of this invention. It is understood that one skilled in theart may be able to make these compounds by similar methods or bycombining other methods known to one skilled in the art. It is alsounderstood that one skilled in the art would be able to make, in asimilar manner as described below, other compounds of structure (I) notspecifically illustrated below by using the appropriate startingcomponents and modifying the parameters of the synthesis as needed. Ingeneral, starting components may be obtained from sources such as SigmaAldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI,and Fluorochem USA, etc. or synthesized according to sources known tothose skilled in the art (see, for example, Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5th edition (Wiley, December2000)) or prepared as described in this invention.

Reaction Scheme I illustrates an exemplary method for preparing anintermediate useful for preparation of compounds of structure (I), whereR¹, L², L³ and M are as defined above, R² and R³ are as defined above orare protected variants thereof and L is an optional linker. Referring toReaction Scheme 1, compounds of structure a can be purchased or preparedby methods well-known to those of ordinary skill in the art. Reaction ofa with M-X, where x is a halogen such as bromo, under Suzuki couplingconditions known in the art results in compounds of structure b.Compounds of structure b can be used for preparation of compounds ofstructure (I) as described below.

Reaction Scheme II illustrates an alternative method for preparation ofintermediates useful for preparation of compounds of structure (I).Referring to reaction Scheme II, where R¹, L¹, L², L³, G and M are asdefined above, and R² and R³ are as defined above or are protectedvariants thereof, a compound of structure c, which can be purchased orprepared by well-known techniques, is reacted with M-G′ to yieldcompounds of structure d. Here, G and G′ represent functional groupshaving complementary reactivity (i.e., functional groups which react toform a covalent bond). G′ may be pendant to M or a part of thestructural backbone of M. G may be any number of functional groupsdescribed herein, such as alkyne.

The compound of structure (I) may be prepared from one of structures bor d by reaction under well-known automated DNA synthesis conditionswith a phosphoramidite compound having the following structure (e):

wherein A is as defined herein and each L is independently an optionallinker.

DNA synthesis methods are well-known in the art. Briefly, two alcoholgroups, for example R² and R³ in intermediates b or d above, arefunctionalized with a dimethoxytrityl (DMT) group and a2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively.The phosphoramidite group is coupled to an alcohol group, typically inthe presence of an activator such as tetrazole, followed by oxidation ofthe phosphorous atom with iodine. The dimethoxytrityl group can beremoved with acid (e.g., chloroacetic acid) to expose the free alcohol,which can be reacted with a phosphoramidite group. The 2-cyanoethylgroup can be removed after oligomerization by treatment with aqueousammonia.

Preparation of the phosphoramidites used in the oligomerization methodsis also well-known in the art. For example, a primary alcohol (e.g., R³)can be protected as a DMT group by reaction with DMT-Cl. A secondaryalcohol (e.g., R²) is then functionalized as a phosphoramidite byreaction with an appropriate reagent such as 2-cyanoethylN,N-dissopropylchlorophosphoramidite. Methods for preparation ofphosphoramidites and their oligomerization are well-known in the art anddescribed in more detail in the examples.

Compounds of structure (I) are prepared by oligomerization ofintermediates b or d and e according to the well-known phophoramiditechemistry described above. The desired number of m and n repeating unitsis incorporated into the molecule by repeating the phosphoramiditecoupling the desired number of times. It will be appreciated thatcompounds of structure (II) as, described below, can be prepared byanalogous methods.

In various other embodiments, compounds useful for preparation of thecompound of structure (I) are provided. The compounds can be preparedabove in monomer, dimer and/or oligomeric form and then the M moietycovalently attached to the compound via any number of syntheticmethodologies (e.g., the “click” reactions described above) to form acompound of structure (I). Accordingly, in various embodiments acompound is provided having the following structure (II):

or a stereoisomer, salt or tautomer thereof, wherein:

A is, at each occurrence, independently a moiety comprising one or more,fused, carbocyclic or heterocyclic ring system;

G is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with a complementary reactivegroup;

L^(1a), L², L³, L⁴ and L⁵ are, at each occurrence, independently anoptional alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, heteroalkynylene or heteroatomic linker;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,—OP(═R_(a))(R_(b))R_(c), Q, a linker comprising a covalent bond to Q, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support or a linker comprising acovalent bond to a further compound of structure (II), wherein: R_(a) isO or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R_(c) is OH, SH, O⁻,S⁻, OR_(d), SR_(d), alkyl, alkoxy, alkylether, alkoxyalkylether,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether; and R_(d) is a counter ion;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with an analyte molecule, asolid support or a complementary reactive group Q;

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and

n is an integer of one or greater.

The G moiety in the compound of structure (II) can be selected from anymoiety comprising a group having the appropriate reactivity group forforming a covalent bond with a complementary group on an M moiety. Inexemplary embodiments, the G moiety can be selected from any of the Qmoieties described herein, including those specific examples provided inTable 1. In some embodiments, G comprises, at each occurrence,independently a moiety suitable for reactions including: the coppercatalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1,3-dipolar cycloaddition), reaction of a diene and dienophile(Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reaction ofa strained alkene with an azide, tetrazine or tetrazole, alkene andazide [3+2] cycloaddition, alkene and tetrazine inverse-demandDiels-Alder, alkene and tetrazole photoreaction and various displacementreactions, such as displacement of a leaving group by nucleophilicattack on an electrophilic atom.

In some embodiments, G is, at each occurrence, independently a moietycomprising an aldehyde, oxime, hydrazone, alkyne, amine, azide,acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide, sulfonylhalide, isothiocyanate, imidoester, activated ester, ketone,α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide,aziridine, tetrazine, tetrazole, phosphine, biotin or thiiranefunctional group.

In other embodiments, G comprises, at each occurrence, independently analkyne or an azide group. In different embodiments, G comprises, at eachoccurrence, independently a reactive group capable of forming afunctional group comprising an alkene, ester, amide, thioester,disulfide, carbocyclic, heterocyclic or heteroaryl group, upon reactionwith the complementary reactive group. For example, in some embodimentthe heteroaryl is triazolyl.

In various other embodiments of the compound of structure (II), L², L³,L⁴ and L⁵ are, at each occurrence, independently C₁-C₆ alkylene, C₂-C₆alkenylene or C₂-C₆ alkynylene.

In other embodiments, the compound has the following structure (IIA):

wherein:

x¹, x², x³, x⁴, x⁵ and x⁶ are, at each occurrence, independently aninteger from 0 to 6.

In other embodiments of structure (II), each L^(1a) is absent. In otherembodiments, each L^(1a) is present, for example L^(1a) is, at eachoccurrence, independently heteroalkylene. In certain embodiments, L^(1a)has the following structure:

In other of any of the foregoing embodiments of compound (II), G is, ateach occurrence, independently

In various embodiments of the compound of structure (IIa), x³ and x⁴ areboth 2 at each occurrence. In other embodiments, x¹, x², x⁵ and x⁶ areeach 1 at each occurrence.

In some other embodiments of the compound of structure (II) or (IIa), Ais at each occurrence, independently a moiety comprising one or more,fused, aryl or heteroaryl ring system. In different embodiments, A is ateach occurrence, independently a moiety comprising one or more, fused,bicyclic or tricyclic, aryl or heteroaryl ring system.

In other more specific embodiments of the compound of structure (II) or(IIa), A is, at each occurrence, independently a fused, carbocyclic orheterocyclic ring system having one of the following structures:

wherein:

a¹, a² and a³ are, at each occurrence, independently a 5, 6 or7-membered carbocyclic or heterocyclic ring; and

L⁶ is a direct bond or a linker.

In yet other embodiments of the compound of structure (II) or (IIa), A,at each occurrence, independently has one of the following structures:

In other embodiments, R⁴ is, at each occurrence, independently OH, O⁻ orOR_(d), and in different embodiments R⁵ is, at each occurrence, oxo.

In some different embodiments of any of the foregoing compounds ofstructure (II) or (IIa), R¹ is H.

In other various embodiments of the compounds of structure (II) or(IIa), R² and R³ are each independently OH or —OP(═R_(a))(R_(b))R_(c).In some different embodiments, R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q.

In still other embodiments of compounds of structure (II) or (IIa), Qis, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with an analyte molecule or asolid support. In other embodiments, Q is, at each occurrence,independently a moiety comprising a reactive group capable of forming acovalent bond with a complementary reactive group Q′. For example, insome embodiments, Q′ is present on a further compound of structure (II)or (IIa) (e.g., in the R² or R³ position), and Q and Q′ comprisecomplementary reactive groups such that reaction of the compound ofstructure (II) or (IIa) and the further compound of structure (II) or(IIa) results in covalently bound dimer of the compound of structure(II) or (IIa). Multimer compounds of structure (II) of (IIa) can also beprepared in an analogous manner and are included within the scope ofembodiments of the invention.

The type of Q group and connectivity of the Q group to the remainder ofthe compound of structure (II) or (IIa) is not limited, provided that Qcomprises a moiety having appropriate reactivity for forming the desiredbond.

In certain embodiments of compounds of structure (II) or (IIa), the Q isa moiety which is not susceptible to hydrolysis under aqueousconditions, but is sufficiently reactive to form a bond with acorresponding group on an analyte molecule or solid support (e.g., anamine, azide or alkyne).

Certain embodiments of compounds of structure (II) or (IIa) comprises Qgroups commonly employed in the field of bioconjugation. For example insome embodiments, Q comprises a nucleophilic reactive group, anelectrophilic reactive group or a cycloaddition reactive group. In somemore specific embodiments, Q comprises a sulfhydryl, disulfide,activated ester, isothiocyanate, azide, alkyne, alkene, diene,dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide,biotin, amino or maleimide functional group. In some embodiments, theactivated ester is an N-succinimide ester, imidoester orpolyflourophenyl ester. In other embodiments, the alkyne is an alkylazide or acyl azide.

Exemplary Q moieties for compounds of structure (II) or (IIa) areprovided in Table I above.

As with compounds of structure (I) or (Ia), in some embodiments ofcompounds of structure (II) or (IIa), wherein Q is SH, the SH moietywill tend to form disulfide bonds with another sulfhydryl group onanother compound of structure (II) or (IIa). Accordingly, someembodiments include compounds of structure (II) or (IIa), which are inthe form of disulfide dimers, the disulfide bond being derived from SH Qgroups.

In some other embodiments of compounds of structure (II) or (IIa), oneof R² or R³ is OH or —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³is a linker comprising a covalent bond to an analyte molecule or alinker comprising a covalent bond to a solid support. For example, insome embodiments the analyte molecule is a nucleic acid, amino acid or apolymer thereof. In other embodiments, the analyte molecule is anenzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer orprion. In still different embodiments, the solid support is a polymericbead or nonpolymeric bead.

In other embodiments of compounds of structure (II) or (IIa), m is, ateach occurrence, independently an integer from 1 to 10. For example, insome embodiments m is, at each occurrence, independently an integer from1 to 5.

In yet different embodiments of compounds of structure (II) or (IIa) nis an integer from 1 to 100. For example, in some embodiments n is aninteger from 1 to 10.

In other different embodiments, the compound of structure (II) isselected from Table 3.

TABLE 3 Exemplary Compounds of Structure (II) No. Structure II-1

The compounds of structure (II) or (IIa) can be used in various methods,for example in embodiments is provided a method for labeling an analytemolecule, the method comprising:

-   -   (a) admixing any of the described compounds of structure (I),        wherein R² or R³ is Q or a linker comprising a covalent bond to        Q, with the analyte molecule;    -   (b) forming a conjugate of the compound and the analyte        molecule; and    -   (c) reacting the conjugate with a compound of formula L^(1b)-G′,        thereby forming at least one covalent bond by reaction of at        least one G and at least one G′,

wherein:

M is a moiety comprising two or more carbon-carbon double bonds and atleast one degree of conjugation;

L^(1b) is an optional alkylene, heteroalkylene or heteroatomic linker;and

G′ is a reactive group complementary to G.

A different embodiment is a method for labeling an analyte molecule, themethod comprising:

-   -   (a) admixing any of the compounds of structure (II) disclosed        herein, wherein R² or R³ is Q or a linker comprising a covalent        bond to Q, with a compound of formula M-L^(1b)-G′, thereby        forming at least one covalent bond by reaction of G and G′; and    -   (b) reacting the product of step (A) with the analyte molecule,        thereby forming a conjugate of the product of step (A) and the        analyte molecule,

wherein:

M is a moiety comprising two or more carbon-carbon double bonds and atleast one degree of conjugation;

L^(1b) is an optional alkylene, heteroalkylene or heteroatomic linker;and

G′ is a reactive group complementary to G.

Further, as noted above, the compound of structure (II) are useful forpreparation of compounds of structure (I). Accordingly, in oneembodiment is provided a method for preparing a compound of structure(I), the method comprising admixing a compound of structure (II) with acompound of formula M-L^(1b)-G′, thereby forming at least one covalentbond by reaction of G and G′, wherein:

M is a moiety comprising two or more carbon-carbon double bonds and atleast one degree of conjugation;

L^(1b) is an optional alkylene, heteroalkylene or heteroatomic linker;and

G′ is a reactive group complementary to G.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES General Methods

¹H NMR spectra were obtained on a JEOL 400 MHz spectrometer. ¹H spectrawere referenced against TMS. Reverse phase HPLC dye analysis wasperformed using a Waters Acquity UHPLC system with a 2.1 mm×50 mmAcquity BEH-C18 column held at 45° C. Mass spectral analysis wasperformed on a Waters/Micromass Quattro micro MS/MS system (in MS onlymode) using MassLynx 4.1 acquisition software. Mobile phase used forLC/MS on dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6mM triethylamine (TEA), pH 8. Phosphoramidites and precursor moleculeswere also analyzed using a Waters Acquity UHPLC system with a 2.1 mm×50mm Acquity BEH-C18 column held at 45° C., employing anacetonitrile/water mobile phase gradient. Molecular weights for monomerintermediates were obtained using tropylium cation infusion enhancedionization on a Waters/Micromass Quattro micro MS/MS system (in MS onlymode). Excitation and emission profiles experiments were recorded on aCary Eclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic acid, pyridine, and THF were purchased fromAldrich. All other chemicals were purchase from Aldrich or TCI and wereused as is with no additional purification.

All oligonucleotide dyes were synthesized on an ABI 394 DNA synthesizerusing standard protocols for the phosphoramidite based couplingapproach. The chain assembly cycle for the synthesis of oligonucleotidephosphoramidates was the following: (i) detritylation, 3%trichloroacetic acid in dichloromethane, 1 min; (ii) coupling, 0.1 Mphosphoramidite and 0.45 M tetrazole in acetonitrile, 10 min; (iii)capping, 0.5 M acetic anhydride in THF/lutidine, 1/1, v/v 15 s; (iv)oxidation, 0.1 M iodine in THF/pyridine/water, 10/10/1, v/v/v, 30 s.

Chemical steps within the cycle were followed by acetonitrile washingand flushing with dry argon for 0.2-0.4 min. Cleavage from the supportand removal of base and phosphoramidate protecting groups was achievedby treatment with ammonia for 1 hour at room temperature.Oligonucleotide dyes were then analyzed by reverse phase HPLC asdescribed above.

Example 1 Synthesis of Phosphoramidite Dye Monomers

In a 250 mL round bottomed flask with stirring bar was placedpyromellitic anhydride (1.0 g, 4.59 mmol) and dioxane (50 mL). To thiswas added ethanolamine (622 μL, 11.5 mmol) and diisopropylethylamine(4.0 mL, 23 mmol). The flask was equipped with a reflux condenser,placed in an oil bath and heated to reflux overnight. The mixture wasallowed to cool. The solids were filtered and retained for later. Thefiltrate was condensed and partitioned between ethyl acetate and water.The ethyl acetate layer was retained and the aqueous layer was extractedtwo additional times with ethyl acetate. The organic layers werecombined, dried over sodium sulfate, filtered and concentrated to asolid. The solids from extraction and the earlier filtration were foundto be identical by TLC and combined to afford the final material. (870mg)

LC/MS of product showed m/z 305 associated with the largest peak.Overall purity was ˜79%.

In a 100 mL round bottomed flask with stirring bar was placed the diol(850 mg, 2.8 mmol) in pyridine (15 mL). The mixture was cooled on iceunder a stream of nitrogen. To this was added 4,4 dimethoxytritylchloride (237 mg, 0.7 mmol). The mixture was stirred at 4° C. overnight.Methanol (2 mL) was added, and the mixture stirred for 15 min beforeconcentrating to a paste on the rotovap. The residue was partitionedbetween a saturated solution of sodium bicarbonate and toluene. Retainedtoluene layer and extracted aqueous layer two additional times withdichloromethane. The organic layers were combined, dried with sodiumsulfate and concentrated. Final purification was accomplished withsilica gel chromatography (dichloromethane/methanol gradient) to affordthe final product as a solid (372 mg).

In a 100 mL round bottomed flask with stirring bar was placed themonoprotected DMTr alcohol (150 mg, 0.24 mmol) in dry dichloromethane(25 mL) with diisopropylethylamine (215 μL, 1.23 mmol).2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (110 μL, 0.49 mmol)was added and the mixture stirred for 30 min at which point TLCindicated the reaction was complete. The mixture was partitioned betweendichloromethane and saturated sodium bicarbonate. The organic layer wasretained, dried over sodium sulfate and concentrated to a yellow oilwhich was used directly on the DNA synthesizer.

In a 250 mL round bottomed flask with stirring bar was placed3,3′,4,4′-biphenyltetracarboxylic dianhydride (1.2 g, 4.1 mmol) anddioxane (80 mL). Ethanolamine (616 μL, 10.2 mmol) anddiisopropylethylamine (3.5 mL) were added, the flask was equipped with areflux condenser and the mixture heated to reflux overnight. Thereaction was cooled and added to a stirring beaker of water (1500 mL) toeffect precipitation. The solids were collected by filtration and driedunder vacuum (0.74 g)

Purity was >95%. Predicted MW is 380.4. MW found was 380.2.

In a 50 mL round bottomed flask with stir bar was placed the diol (700mg, 1.8 mmol) in pyridine (9 mL). The mixture was cooled on ice undernitrogen. To this was added 4,4 dimethoxytrityl chloride (468 mg, 1.4mmol). The mixture was stirred at 4° C. overnight. Methanol (2 mL) wasadded, and the mixture stirred for 10 min before concentrating to apaste on the rotovap. Final purification was accomplished with silicagel chromatography (dichloromethane/methanol gradient) to afford thefinal product as a solid (151 mg).

Overall purity is ˜89%. M/Z 773.8 is consistent with product+tropylium.

In a 100 mL round bottomed flask with stirring bar was placed themonoprotected DMTr alcohol (132 mg, 0.19 mmol) in dry dichloromethane(25 mL) with diisopropylethylamine (168 μL, 0.74 mmol).2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (86 μL, 0.38 mmol) wasadded and the mixture stirred for 30 min at which point TLC indicatedthe reaction was complete. The mixture was partitioned betweendichloromethane and saturated sodium bicarbonate. The organic layer wasretained, dried over sodium sulfate and concentrated to an oil which wasused directly on the DNA synthesizer.

In a 250 mL round bottomed flask with stirring bar was placed4,4′-Oxydiphthalic anhydride (1.2 g, 3.9 mmol) and dioxane (90 mL).Ethanolamine (584 μL, 9.7 mmol) and diisopropylethylamine (3.4 mL) wereadded, the flask was equipped with a reflux condenser and the mixtureheated to reflux overnight. The reaction was cooled and concentrated ona rotovap to a paste which was partitioned between ethyl acetate andwater. The organic layer was retained and the aqueous layer wasextracted and additional time with ethyl acetate. The organic layerswere combined, dried over sodium sulfate and concentrated to the finalsolid product (1.33 g)

Overall purity is ˜83%. Main peak at 1.16 min. Calculated MW is 396.4.MW found is 396.2.

In a 20 mL round bottomed flask with stir bar was placed the diol (700mg, 1.8 mmol) in pyridine (9 mL). The mixture was cooled on ice undernitrogen. To this was added 4,4 dimethoxytrityl chloride (449 mg, 1.3mmol). The mixture was stirred at 4° C. overnight. Methanol (2 mL) wasadded, and the mixture stirred for 15 min before concentrating to apaste on the rotovap. Final purification was accomplished with silicagel chromatography (dichloromethane/methanol gradient) to afford thefinal product as a solid (600 mg).

Overall purity is ˜82%. Predicted MW is 698.7. MW found is 788.5, whichis consistent with product+tropylium.

In a 100 mL round bottomed flask with stirring bar was placed themonoprotected DMTr alcohol (100 mg, 0.14 mmol) in dry dichloromethane(25 mL) with diisopropylethylamine (125 μL, 0.71 mmol).2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (64 μL, 0.28 mmol) wasadded and the mixture stirred for 30 min at which point TLC indicatedthe reaction was complete. The mixture was partitioned betweendichloromethane and saturated sodium bicarbonate. The organic layer wasretained, dried over sodium sulfate and concentrated to a yellow oilwhich was used directly in solid phase oligonucleotide synthesis.

In a 250 mL round bottomed flask with stirring bar was placed4,4′-(4,4′-Isopropylidinediphenoxy)bis(phthalic anhydride) (1.5 g, 2.9mmol) and dioxane (50 mL). Ethanolamine (435 μL, 7.2 mmol) anddiisopropylethylamine (2.5 mL) were added, the flask was equipped with areflux condenser and the mixture heated to reflux overnight. Thereaction was cooled and concentrated on a rotovap to a paste which waspartitioned between dichloromethane and water. The organic layer wasretained and the aqueous layer was extracted two additional times withdichloromethane. The organic layers were combined, dried over sodiumsulfate and concentrated to the final solid product (400 mg).

Overall purity is ˜89%. Predicted MW is 606.6. MW found is 606.4.

In a 20 mL round bottomed flask with stir bar was placed the diol (400mg, 0.7 mmol) in pyridine (3 mL). The mixture was cooled on ice undernitrogen. To this was added 4,4 dimethoxytrityl chloride (168 mg, 0.5mmol). The mixture was stirred at 4° C. overnight. Methanol (1 mL) wasadded, and the mixture stirred for 10 min before concentrating to apaste on the rotovap. Final purification was accomplished with silicagel chromatography (dichloromethane/methanol gradient) to afford thefinal product as a solid. Overall purity is ˜36%. M/Z at 1000 isconsistent with mass+tropylium)

In a 250 mL round bottomed flask with stirring bar was placedpyromellitic dianhydride (0.75 g, 3.4 mmol) and dioxane (80 mL). SerineMethyl ester (1.2 g, 7.6 mmol) and diisopropylethylamine (4.8 mL) wereadded, the flask was equipped with a reflux condenser and the mixtureheated to reflux overnight. The reaction was cooled and concentrated ona rotovap to a paste which was partitioned between ethyl acetate andcitric acid (500 mM). The organic layer was retained and the aqueouslayer was extracted and two additional times with ethyl acetate. Theorganic layers were combined, dried over sodium sulfate and concentratedto the final oil (1.4 g).

Calculated MW is 420.33. MW found is 420.1.

In a 100 mL round bottomed flask with stir bar was placed the diol (1.4g, 3.5 mmol) in pyridine (17 mL). The mixture was cooled on ice undernitrogen. To this was added 4,4 dimethoxytrityl chloride (883 mg, 2.6mmol). The mixture was stirred at 4° C. overnight. Methanol (5 mL) wasadded, and the mixture stirred for 10 min before concentrating to apaste on the rotovap. The residue was partitioned between a saturatedsolution of sodium bicarbonate and ethyl acetate. Retained organic layerand extracted aqueous layer two additional times with ethyl acetate. Theorganic layers were combined, dried with sodium sulfate andconcentrated. Final purification was accomplished with silica gelchromatography (dichloromethane/ethyl acetate gradient) to afford thefinal product as an oil (1.7 g). MW at 812 consistent with mass plustropyllium.

Example 2 Synthesis of Oligomer Dyes

Oligomer dyes were synthesized on an Applied Biosystems 394 DNA/RNAsynthesizer or on GE AKTÅ 10 OligoPilot on either 1 μmol or 10 μmolscales and possessed a 3′-phosphate group. Dyes were synthesizeddirectly on CPG beads or on polystyrene solid support. The dyes weresynthesized in the 3′ to 5′ direction by standard solid phase DNAmethods. Coupling methods employed standard β-cyanoethyl phosphoramiditechemistry conditions. Different number of repeating units wereincorporated by repeating the synthesis cycle the desired number oftimes with an appropriate phosphoramidite. All phosphoramidite monomerswere dissolved in acetonitrile/dichloromethane (0.1 M solutions), andwere added in successive order using the following synthesis cycles: 1)removal of the 5′-dimethoxytrityl protecting group with dichloroaceticacid in toluene, 2) coupling of the next phosphoramidite with activatorreagent in acetonitrile, 3) oxidation with iodine/pyridine/water, and 4)capping with acetic anhydride/1-methylimidizole/acetonitrile. Thesynthesis cycle was repeated until the 5′ Oligofloroside was assembled.At the end of the chain assembly, the monomethoxytrityl (MMT) group ordimthoxytrityl (DMT) group was removed with dichloroacetic acid indichloromethane or dichloroacetic acid in toluene.

The dyes were cleaved from the solid support and deprotected as follows:

A 1 mL micropipettor was used to add 4504, of concentrated NH₄OH to ˜25mg of reacted CPG solid support in a 1.5 mL Eppendorf tube. The slurrywas mixed briefly using a Vortex mixer and allowed to settle beforeplacing (open) on a 55° C. heating block until gas formation (andbubbling) started to diminish, at which point the tube was tightlyclosed. Heat treatment was for 2 hours (+/−15 minutes) and tubes werethen removed to cool to room temperature. The tube and its contents werespun in a centrifuge at its maximum speed (13400 rpm) for 1 minute, andthen the supernatant was removed with a glass pipette and placed into asecond, labeled, 1.5 mL Eppendorf tube, taking care not to include thesupport. The support was washed and spun-down 2× with ˜150 μL ofacetonitrile to help maximize dye removal, and the washings werecarefully removed from support and added to the labeled secondary tubes.Clarified supernatant was dried completely in a CentriVap concentratorat 40° C. to remove NH₄OH.

Example 3 Characterization of Oligomer Dyes

1 mL of deionized water was added to the dried dye sequence preparedaccording to Example 2 to re-constitute and establish a concentratedstock of ˜0.3 to 1.0 mM (determined later). 24, aliquots of each dyeconstruct were analyzed by HPLC-MS to determine identity and relativepurity using 45° C. heated ultra-high performance 2.1 mm×50 mm C18column (1.7 μm) with 150 mM HFIP/TEA (pH9) mobile phase, and methanol asorganic elution component. Gradient was from 1-100% over 10 minutes.Electrospray ionization was used (in negative mode) to determine themolecular weights of the dye sequences and help to characterizeimpurities.

A sample was taken from a concentrated stock using a micropipettor anddiluted appropriately in 0.1×PBS (10× to 100×) to be within linear rangeof the NanoDrop UV-vis spetrophotomer (Thermo Scientific). A blankmeasurement was performed on the NanoDrop using 0.1×PBS, and then theabsorbance of the diluted dye sequence at an appropriate wavelength wasrecorded. Extinction coefficients (c) were determined by the totalnumber of fluors (M moieties) in the dye construct, using 75,000 M⁻¹cm⁻¹ for each fluorescein (F; read at 494 nm); 34,500 for each pyrene(Y; read at 343 nm); and 40,000 for each perylene (E; read at 440 nm)present in the sequence. Spacers and linkers are presumed to have noeffect on ε.

With concentration determined, the dye stock was diluted in the NaPO₄(0.1 M at pH 7.5) and NaCO₃ (0.1 M at pH 9.0) buffers to make solutionsof 2 μM (or 5 μM, whatever works with the linear range of theinstrument) at a final volume of ˜3.5 mL. These solutions were scannedby UV/Vis, and then used them to make a second dilution in theappropriate buffer for reading on the fluorimeter, in the range of 10-50nM. The necessary concentration will vary depending upon the identity ofthe M moiety.

Using a 1 cm quartz cuvette, the absorbance of the 2 μM sample wasdetermined, scanning from 300 nm to 700 nm. Scan speed was set tomedium.

Using a 1 cm quartz cuvette and a Cary Eclipse spectrometer, theemission of the 25 nM sample was read using an appropriate excitationwavelength (494 nm for above dye) and scanning from 499 nm to 700 nm.Scan speed was set to medium.

The UV absorbance (FIG. 1) and fluorescence emission (FIG. 2) spectra ofcompounds I-1, 1-2, and 1-3 were compared to a “monomeric” fluoresceincompound (compound A). As expected, the UV absorbance is approximately 2times greater for compounds I-1, 1-2 and 1-3, relative to Compound A.Further, it was found that when m=1 (Compound I-1), the emission wasonly slightly higher than the emission for compound A. However, when m=2or more (Compounds 1-2 and 1-3), the emission is approximately twice theamount relative to Compound A, indicating little or no quenching of thechromophore. Accordingly, the rigid linker A appears to preventquenching of the fluorophores, especially as the number of A moietiesincreases (i.e., as m increases).

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound having the following structure (I):

or a stereoisomer, salt or tautomer thereof, wherein: A is, at eachoccurrence, independently a moiety comprising one or more, fused,carbocyclic or heterocyclic ring system; M is, at each occurrence,independently a moiety comprising two or more carbon-carbon double bondsand at least one degree of conjugation; L¹ is at each occurrence,independently either: i) an optional alkylene, alkenylene, alkynylene,heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomiclinker; or ii) a linker comprising a functional group capable offormation by reaction of two complementary reactive groups; L², L³, L⁴and L⁵ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker; R¹ is, at each occurrence,independently H, alkyl or alkoxy; R² and R³ are each independently H,OH, SH, alkyl, alkoxy, alkylether, —OP(═R_(a))(R_(b))R_(c), Q, a linkercomprising a covalent bond to Q, a linker comprising a covalent bond toan analyte molecule, a linker comprising a covalent bond to a solidsupport or a linker comprising a covalent bond to a further compound ofstructure (I), wherein: R_(a) is O or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d)or SR_(d); R_(c) is OH, SH, O⁻, S⁻, OR_(d), SR_(d), alkyl, alkoxy,alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether; and R_(d)is a counter ion; R⁴ is, at each occurrence, independently OH, SH, O⁻,S⁻, OR_(d) or SR_(d); R⁵ is, at each occurrence, independently oxo,thioxo or absent; Q is, at each occurrence, independently a moietycomprising a reactive group capable of forming a covalent bond with ananalyte molecule, a solid support or a complementary reactive group Q; mis, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and n is an integer of one or greater. 2-4. (canceled)
 5. Thecompound of claim 1, wherein A, at each occurrence, independently hasone of the following structures:

6-10. (canceled)
 11. The compound of claim 61, wherein for at least oneoccurrence of L¹, L¹-M has the following structure:

wherein L^(1a) and L^(1b) are each independently optional linkers. 12.The compound of claim 61, wherein for at least one occurrence of L¹,L¹-M has the following structure:

wherein L^(1a) and L^(1b) are each independently optional linkers.13-16. (canceled)
 17. The compound of claim 1, wherein L¹ is at eachoccurrence, independently an optional alkylene or heteroalkylene linker.18. (canceled)
 19. The compound of claim 1, wherein the compound has thefollowing structure (IA):

wherein: x¹ x², x³, x⁴, x⁵ and x⁶ are, at each occurrence, independentlyan integer from 0 to
 6. 20-23. (canceled)
 24. The compound of claim 1,wherein R⁴ is, at each occurrence, independently OH, O⁻ or OR_(d). 25.The compound of claim 1, wherein R⁵ is, at each occurrence, oxo.
 26. Thecompound of claim 1, wherein R¹ is H.
 27. The compound of claim 1,wherein R² and R³ are each independently OH or —OP(═R_(a))(R_(b))R_(c).28. The compound of claim 1, wherein one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q. 29-32. (canceled)
 33. The compound ofclaim 1, wherein Q is a moiety selected from Table
 1. 34. The compoundof claim 1, wherein one of R² or R³ is OH or —OP(═R_(a))(R_(b))R_(c),and the other of R² or R³ is a linker comprising a covalent bond to ananalyte molecule or a linker comprising a covalent bond to a solidsupport wherein the analyte molecule is a nucleic acid or polymerthereof, an amino acid or polymer thereof, an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion and the solid supportis a polymeric bead or nonpolymeric bead. 35-45. (canceled)
 46. Thecompound of claim 1, wherein M is, at each occurrence, independently adimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY,his-fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin,benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9, 10-ethynylanthracene or ter-naphthyl moiety.47. The compound of claim 1, wherein M is, at each occurrence,independently p-terphenyl, perylene, azobenzene, phenazine,phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene,cyanine, perylene imide, or perylene amide or derivative thereof. 48.The compound of claim 1, wherein M is, at each occurrence, independentlya coumarin dye, resorufin dye, dipyrrometheneboron difluoride dye,ruthenium bipyridyl dye, energy transfer dye, thiazole orange dye,polymethine or N-aryl-1,8-naphthalimide dye.
 49. The compound of claim1, wherein M is, at each occurrence, independently pyrene, perylene,perylene monoimide or 6-FAM or derivative thereof.
 50. The compound ofclaim 1, wherein M, at each occurrence, independently has one of thefollowing structures:


51. The compound of claim 1, wherein the compound is selected from Table2. 52-57. (canceled)
 58. A method for visually detecting an analytemolecule, the method comprising: (a) admixing the compound of claim 1,wherein R² or R³ is Q or a linker comprising a covalent bond to Q, withthe analyte molecule; (b) forming a conjugate of the compound and theanalyte molecule; and (c) detecting the conjugate by its visibleproperties. 59-60. (canceled)
 61. A compound having the followingstructure (II):

or a stereoisomer, salt or tautomer thereof, wherein: A is, at eachoccurrence, independently a moiety comprising one or more, fused,carbocyclic or heterocyclic ring system; G is, at each occurrence,independently a moiety comprising a reactive group capable of forming acovalent bond with a complementary reactive group; L^(1a), L², L³, L⁴and L⁵ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker; R¹ is, at each occurrence,independently H, alkyl or alkoxy; R² and R³ are each independently H,OH, SH, alkyl, alkoxy, alkylether, —OP(═R_(a))(R_(b))R_(c), Q, a linkercomprising a covalent bond to Q, a linker comprising a covalent bond toan analyte molecule, a linker comprising a covalent bond to a solidsupport or a linker comprising a covalent bond to a further compound ofstructure (II), wherein: R_(a) is O or S; R_(b) is OH, SH, O⁻, S⁻,OR_(d) or SR_(d); R_(c) is OH, SH, O⁻, S⁻, OR_(d), SR_(d), alkyl,alkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether; and R_(d) is a counter ion; R⁴ is, at eachoccurrence, independently OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R⁵ is, ateach occurrence, independently oxo, thioxo or absent; Q is, at eachoccurrence, independently a moiety comprising a reactive group capableof forming a covalent bond with an analyte molecule, a solid support ora complementary reactive group Q; m is, at each occurrence,independently an integer of zero or greater, provided that at least oneoccurrence of m is an integer of one or greater; and n is an integer ofone or greater. 62-97. (canceled)